1. Field of the Invention
This invention relates to compositions, methods and kits for determining an analyte in a sample.
Chemiluminescent labels for immunoassays and nucleic acid probe assays provide a high degree of sensitivity when compared to other commonly used labels. An excellent overview of the subject is discussed in McCapra, et al., Journal of Bioluminescence and Chemiluminescence 4:51-58 (1989).
Particularly important chemiluminescent compounds are acridinium esters and amides, which must be stored in water at a pH that is compatible with protein stability requirements. This results in the formation of a pseudobase, which is not chemiluminescent. Chemiluminescence is initiated by adding acid to form the acridinium salt, followed by the addition of strongly alkaline hydrogen peroxide. In general, these conditions are not ideal for immunoassays as they are not suitable for preserving receptor-ligand binding and are undesirably complex.
The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of materials (analytes) that may be readily and accurately determined, as well as the methods for the determination. Convenient, reliable and non-hazardous means for detecting the presence of low concentrations of materials in liquids is desired. In clinical chemistry these materials may be present in body fluids in concentrations below 10.sup.-12 molar. The difficulty of detecting low concentrations of these materials is increased by the relatively small sample sizes that can be utilized.
In developing an assay there are many considerations. One consideration is the signal response to changes in the concentration of analyte. A second consideration is the ease with which the protocol for the assay may be carried out. A third consideration is the variation in interference from sample to sample. Ease of preparation and purification of the reagents, availability of equipment, ease of automation and interaction with material of interest are some of the additional considerations in developing a useful assay.
One broad category of techniques involves the use of a receptor which can specifically bind to a particular spacial and polar organization of a labeled ligand as a function of the presence of the analyte. The observed effect of binding by the receptor will depend upon the label. In some instances the binding of the receptor merely provides for a differentiation in molecular weight between bound and unbound labeled ligand. In other instances the binding of the receptor will facilitate separation of bound labeled ligand from free labeled ligand or it may affect the nature of the signal obtained from the label so that the signal varies with the amount of receptor bound to labeled ligand. A further variation is that the receptor is labeled and the ligand unlabeled. Alternatively, both the receptor and ligand are labeled or different receptors are labeled with different labels where the labels interact when in close proximity and the amount of ligand present affects the degree to which the labels of the receptor may interact.
There is a continuing need for new and accurate techniques that can be adapted for a wide spectrum of different ligands or be used in specific cases where other methods may not be readily adaptable.
Homogeneous immunoassays have previously been described for small molecules. These assays include Syva Company's FRAT.RTM. assay, EMIT.RTM. assay, enzyme channeling immunoassay, and fluorescence energy transfer immunoassay (FETI); enzyme inhibitor immunoassays (Hoffman LaRoche and Abbott Laboratories); and fluorescence polarization immunoassay (Dandlicker), among others. All of these methods have limited sensitivity, and only a few including FETI and enzyme channeling, are suitable for large multiepitopic analytes.
Chemiluminescent compounds find wide application in the assay field because of their ability to emit light. For this reason, luminescers have been utilized as labels in assays such as nucleic acid assays and immunoassays. For example, a member of a specific binding pair is conjugated to a luminescer and various protocols are employed. The luminescer conjugate can be partitioned between a solid phase and a liquid phase in relation to the amount of analyte in a sample suspected of containing the analyte. By measuring the luminescence of either of the phases, one can relate the level of luminescence observed to a concentration of the analyte in the sample.
Chemiluminescent labels have been described for immunoassays and nucleic acid assays where a group, which is covalently bound to a binding partner, on chemical activation emits light. A nucleic acid assay kit utilizing an acridinium ester is sold by Genprobe (Pace 2 system.RTM., San Diego, Calif.) and MagicLite.RTM. immunoassay kits using this type of label are sold by Ciba-Geigy (Basel, Switzerland).
Although, chemiluminescent labels have the advantage of offering exceptional sensitivity in ligand binding assays, one or more chemical activation steps are usually needed. Therefore, there is a need for novel chemiluminescent compounds that do not require several activation steps.
2. Brief Description of the Related Art
U.S. Pat. No. 3,876,659 discloses novel spiro tricyclic isoindolines.
U.S. Pat. Nos. 4,380,580 and 4,383,031, respectively, describe heterogeneous and homogeneous chemiluminescent specific binding assays.
U.S. Pat. No. 4,891,324 describes the use of a particle with a luminescer in assays.
European Patent Application 0 270 946 A2 pertains to chromogenic acridinone enzyme substrates useful in the detection of enzymes.
European Patent Application 0 322 926 A2 describes assays utilizing improved chemiluminescent esters, thioesters, and amides.
European Patent Application 0 324 202 A1 discloses acridinium compounds as chemiluminogenic labels.
European Patent Application 0 421 788 A2 describes a haloperoxidase-acid-optimum chemiluminescence assay system for determining the presence or amount of an analyte in a liquid sample. The system utilizes haloperoxidase, a halide, an oxidant and a chemiluminigenic substrate.
PCT 88/00695 describes the use of dioxetanes in assays, where the dioxetanes contain an enzyme-cleavable group, the removal of which results in a negatively charged substituent being bonded to the dioxetane. This causes the dioxetane to decompose to form a luminescent substance.
Heller, et al. describe chemiluminescent and fluorescent probes for DNA hybridization systems in "Rapid Detection and Identification of Infectious Agents" Academic Press, Inc., pages 245-257 (1985).
Zomer, et al. describe chemiluminogenic labels in Analytica Chimica Acta 227:11-19 (1989).